Insoluble Oat Spelt Xylan Research Articles

The extracellular endo-\u03b2-1,4-xylanase with multidomain from the extreme thermophile Caldicellulosiruptor lactoaceticus is specific for insoluble xylan degradation

BackgroundThe extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus can degrade and metabolize untreated lignocellulosic biomass containing xylan. The mechanism of the bacterium for degradation of insoluble xylan in untreated biomass has not been revealed.ResultsIn the present study, the only annotated extracellular endo-β-1,4-xylanase (Xyn10B) with multidomain structures in C. lactoaceticus genome was biochemically characterized. Xyn10B contains three N-terminal consecutive family 22 carbohydrate-binding modules (CBMs), one GH10 catalytic domain (CD), two family 9 CBMs and two S-layer homology (SLH) modules in the C-terminal. CBM22a shares 27.1% and 27.2% sequence homology with CBM22b and CBM22c, respectively. The sequence homology between two CBM9 s and two SLHs is 26.8% and 25.6%, respectively. To elucidate the effect of multiple domains on the enzymatic properties of Xyn10B, the truncated variants of which (Xyn10B-TM1: CBM22a-CBM22b-CBM22c-CD10; Xyn10B-TM2: CBM22c-CD10; Xyn10B-TM3: CBM22c-CD10-CBM9a; and Xyn10B-TM4: CD10-CBM9a) were separately reconstructed, recombinantly expressed and biochemically characterized. Enzymatic properties studies showed that the optimal temperature for all four Xyn10B truncations was 65 °C. Compared to Xyn10B-TM3 and Xyn10B-TM4, Xyn10B-TM1 and Xyn10B-TM2 had higher hydrolytic activity, thermostability and affinity on insoluble substrates. It is noteworthy that Xyn10B-TM1 and Xyn10B-TM2 have higher enzymatic activity on insoluble xylan than the soluble counterparts, whereas Xyn10B-TM3 and Xyn10B-TM4 showed opposite characteristics. The kinetic parameters analysis of Xyn10B-TM1 on xylan showed Vmax was 5740, 1300, 1033, and 3925 U/μmol on insoluble oat spelt xylan (OSX), soluble beechwood xylan (BWX), soluble sugar cane xylan (SCX), and soluble corncob xylan (CCX), respectively. The results indicated that CBM22s especially CBM22c promoted the hydrolytic activity, thermostability and affinity on insoluble substrates of the Xyn10B truncations. The functions of CBM22, CBM9, CD and SLH are different, while contribute synergetically to the thermostability, protein structure integrity, substrate binding, and high hydrolytic activity on insoluble xylan of untreated lignocellulosic biomass. The domains of CBM22, CBM9, CD and SLH have different characteristics, which synergistically promote the thermostability, protein structure integrity, affinity on insoluble substrates and enzymatic activity properties of Xyn10B.ConclusionsThe extracellular endo-β-1,4-xylanase with multidomain structures of CBM, CD and SLH promote the biodegradation of insoluble xylan in untreated lignocellulosic biomass by thermophilic C. lactoaceticus.

Xylan degradation by the human gut Bacteroides xylanisolvens XB1A(T) involves two distinct gene clusters that are linked at the transcriptional level.

BackgroundPlant cell wall (PCW) polysaccharides and especially xylans constitute an important part of human diet. Xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. Xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. In order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont Bacteroides xylanisolvens XB1AT.ResultsTranscriptomic analyses of B. xylanisolvens XB1AT grown on insoluble oat-spelt xylan (OSX) at mid- and late-log phases highlighted genes in a polysaccharide utilization locus (PUL), hereafter called PUL 43, and genes in a fragmentary remnant of another PUL, hereafter referred to as rPUL 70, which were highly overexpressed on OSX relative to glucose. Proteomic analyses supported the up-regulation of several genes belonging to PUL 43 and showed the important over-production of a CBM4-containing GH10 endo-xylanase. We also show that PUL 43 is organized in two operons and that the knockout of the PUL 43 sensor/regulator HTCS gene blocked the growth of the mutant on insoluble OSX and soluble wheat arabinoxylan (WAX). The mutation not only repressed gene expression in the PUL 43 operons but also repressed gene expression in rPUL 70.ConclusionThis study shows that xylan degradation by B. xylanisolvens XB1AT is orchestrated by one PUL and one PUL remnant that are linked at the transcriptional level. Coupled to studies on other xylanolytic Bacteroides species, our data emphasize the importance of one peculiar CBM4-containing GH10 endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. Our results also suggest that B. xylanisolvens XB1AT has specialized in the degradation of xylans of low complexity. This functional feature may provide a niche to all xylanolytic bacteria harboring similar PULs. Further functional and ecological studies on fibrolytic Bacteroides species are needed to better understand their role in dietary fiber degradation and their impact on intestinal health.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2680-8) contains supplementary material, which is available to authorized users.

Studies on properties of the xylan‑binding domain and linker sequence of xylanase XynG1‑1 from Paenibacillus campinasensis G1‑1.

Xylanase XynG1-1 from Paenibacillus campinasensis G1-1 consists of a catalytic domain (CD), a family 6_36 carbohydrate-binding module which is a xylan-binding domain (XBD), and a linker sequence (LS)between them. The structure of XynG1-3 from Bacillus pumilus G1-3 consists only of a CD. To investigate the functions and properties of the XBD and LS of XynG1-1, two truncated forms (XynG1-1CDL, XynG1-1CD) and three fusion derivatives (XynG1-3CDL, XynG1-3CDX and XynG1-3CDLX) were constructed and biochemically characterized. The optimum conditions for the catalytic activity of mutants of XynG1-1 and XynG1-3 were 60 °C and pH 7.0, and 55 °C and pH 8.0, respectively, the same as for the corresponding wild-type enzymes. XynGs with an XBD were stable over a broad temperature (30-80 °C)and pH range (4.0-11.0), respectively, on incubation for 3 h. Kinetic parameters (Km, kcat, kcat/Km) of XynGs were determined with soluble birchwood xylan and insoluble oat spelt xylan as substrates. XynGs with the XBD showed better affinities toward, and more efficient catalysis of hydrolysis of the insoluble substrate. The XBD had positive effects on thermostability and pH stability and a crucial function in the ability of the enzyme to bind and hydrolyze insoluble substrate. The LS had little effect on the overall stability of the xylanase and no relationship with affinities for soluble and insoluble substrates or catalytic efficiency.

Fusion of a xylan-binding module to gluco-oligosaccharide oxidase increases activity and promotes stable immobilization.

The xylan-binding module Clostridium thermocellum CBM22A was successfully fused to a gluco-oligosaccharide oxidase, GOOX-VN, from Sarocladium strictum via a short TP linker, allowing the fused protein to effectively bind different xylans. The presence of the CtCBM22A at the N-terminal of GOOX-VN increased catalytic activity on mono- and oligo-saccharides by 2-3 fold while not affecting binding affinity to these substrates. Notably, both GOOX-VN and its CBM fusion also showed oxidation of xylo-oligosaccharides with degrees of polymerization greater than six. Whereas fusion to CtCBM22A did not alter the thermostability of GOOX-VN or reduce substrate inhibition, CtCBM22A_GOOX-VN could be immobilized to insoluble oat spelt xylan while retaining wild-type activity. QCM-D analysis showed that the fused enzyme remained bound during oxidation. These features could be harnessed to generate hemicellulose-based biosensors that detect and quantify the presence of different oligosaccharides.

The carbohydrate-binding module of xylanase from Nonomuraea flexuosa decreases its non-productive adsorption on lignin

BackgroundThe enzymatic hydrolysis step converting lignocellulosic materials into fermentable sugars is recognized as one of the major limiting steps in biomass-to-ethanol process due to the low efficiency of enzymes and their cost. Xylanases have been found to be important in the improvement of the hydrolysis of cellulose due to the close interaction of cellulose and xylan. In this work, the effects of carbohydrate-binding module (CBM family II) of the xylanase 11 from Nonomuraea flexuosa (Nf Xyn11) on the adsorption and hydrolytic efficiency toward isolated xylan and lignocellulosic materials were investigated.ResultsThe intact family 11 xylanase of N. flexuosa clearly adsorbed on wheat straw and lignin, following the Langmuir-type isotherm. The presence of the CBM in the xylanase increased the adsorption and hydrolytic efficiency on insoluble oat spelt xylan. But the presence of the CBM did not increase adsorption on pretreated wheat straw or isolated lignin. On the contrary, the CBM decreased the adsorption of the core protein to lignin containing substrates, indicating that the CBM of N. flexuosa xylanase did not contribute to the non-productive adsorption.ConclusionThe CBM of the N. flexuosa xylanase was shown to be a xylan-binding module, which had low affinity on cellulose. The CBM of the N. flexuosa xylanase reduced the non-specific adsorption of the core protein to lignin and showed potential for improving the hydrolysis of lignocellulosic materials to platform sugars.

Xylans inhibit enzymatic hydrolysis of lignocellulosic materials by cellulases

Hemicelluloses have been found to be physical barriers in the hydrolysis of cellulose, and prevent the access of enzymes to cellulose surface. In addition, soluble hemicelluloses may strongly inhibit the cellulase activity. In this work, birchwood xylan clearly inhibited the enzymatic hydrolysis of wheat straw, Avicel and nanocellulose by cellulases. Hydrolysis efficiencies of cellobiohydrolase I (CBHI, from Thermoascus aurantiacus), cellobiohydrolase II (CBHII, from Trichoderma reesei) and endoglucanase II (from T. aurantiacus) were clearly inhibited by birchwood xylan, respectively. The strongest inhibitory effect of birchwood xylan was observed on the hydrolysis of Avicel by CBHI and CBHII, as a dramatically decreased formation of the main product, cellobiose. After additions of soluble and insoluble oat spelt xylan, cleaved cellobiose units by CBHI from cellulose chain decreased from 8 to 4 and 6, respectively. The results in this work demonstrated that xylans clearly inhibited the hydrolysis efficiencies of both endoglucanase and cellobiohydrolase.

Cloning and characterization of a modular GH5 β-1,4-mannanase with high specific activity from the fibrolytic bacterium Cellulosimicrobium sp. strain HY-13

The gene (1272-bp) encoding a β-1,4-mannanase from a gut bacterium of Eisenia fetida, Cellulosimicrobium sp. strain HY-13 was cloned and expressed in Escherichia coli. The recombinant β-1,4-mannanase (rManH) was approximately 44.0kDa and has a catalytic GH5 domain that is 65% identical to that of the Micromonospora sp. β-1,4-mannosidase. The enzyme exhibited the highest catalytic activity toward mannans at 50°C and pH 6.0. rManH displayed a high specific activity of 14,711 and 8498IUmg−1 towards ivory nut mannan and locust bean gum, respectively; however it could not degrade the structurally unrelated polysaccharides, mannobiose, or p-nitrophenyl sugar derivatives. rManH was strongly bound to ivory nut mannan, Avicel, chitosan, and chitin but did not attach to curdlan, insoluble oat spelt xylan, lignin, or poly(3-hydroxybutyrate). The superior biocatalytic properties of rManH suggest that the enzyme can be exploited as an effective additive in the animal feed industry.

Both Substrate Hydrolysis and Secondary Substrate Binding Determine Xylanase Mobility as Assessed by FRAP

Xylanases (EC 3.2.1.8) are enzymes that can hydrolyze the xylan backbone internally. Therefore, they are important for biomass breakdown and they are also often added in various biotechnological applications. In this study, the relationship between their substrate binding affinity and hydrolysis, on the one hand, and their movement over natural substrates, on the other hand, was investigated. Fluorescence recovery after photobleaching (FRAP) experiments using different Bacillus subtilis xylanase A (XBS) mutants were conducted on water-unextractable wheat flour arabinoxylan (WU-AX) and insoluble oat spelt xylan (OSX). To assess the importance of substrate hydrolysis, FRAP of a catalytically inactive mutant was compared to that of the wild-type enzyme. For the wild-type enzyme, substrate binding and a complete recovery of fluorescence after photobleaching was observed on both substrates. For the inactive mutant, however, substrate binding but no fluorescence recovery was observed on WU-AX, while very slow recovery was observed on OSX. Furthermore, the importance of substrate binding to a secondary xylan binding site (SBS) for enzyme mobility was studied by testing two mutants with a modified SBS (N54W-N141Q and G56A-T183A-W185A) that showed different behavior on the tested substrates. On OSX, the two modified enzymes both showed higher mobility than the wild-type enzyme. On WU-AX, in contrast, the N54W-N141Q mutant displayed a lower mobility than the wild-type enzyme, while the G56A-T183A-W185A mutant showed higher mobility. The results clearly demonstrate that both substrate hydrolysis and substrate targeting are key factors for XBS mobility.

A highly active endo-β-1,4-mannanase produced by Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bacterium in the gut of Eisenia fetida

A xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum, guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManK, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50 °C and pH 7.0, ManK exhibited extraordinary high specific activities of 7109 IU/mg and 5158 IU/mg toward locust bean gum and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. Thin layer chromatography revealed that the enzyme degraded locust bean gum to mannobiose and mannotetraose. No detectable amount of mannose was produced from hydrolytic reactions with the substrates. ManK strongly attached to Avicel, β-cyclodextrin, lignin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManK suggest that it is a unique endo-β-1,4-mannanase without additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.

Purification and characterization of a high-thermostable β-xylanase from newly isolated Thermomyces lanuginosus THKU-49

Highly thermostable β-xylanase produced by newly isolated Thermomyces lanuginosus THKU-49 strain was purified in a four-step procedure involving ammonium sulfate precipitation and subsequent separation on a DEAE-Sepharose fast flow column, hydroxylapatite column, and Sephadex G-100 column, respectively. The enzyme purified to homogeneity had a specific activity of 552U/mg protein and a molecular weight of 24.9 kDa. The optimal temperature of the purified xylanase was 70°C, and it was stable at temperatures up to 60°C at pH 6.0; the optimal pH was 5.0–7.0, and it was stable in the pH range 3.5–8.0 at 4°C. Xylanase activity was inhibited by Mn2+, Sn2+, and ethylenediaminetetraacetic acid. The xylanase showed a high activity towards soluble oat spelt xylan, but it exhibited low activity towards insoluble oat spelt xylan; no activity was found to carboxymethylcellulose, avicel, filter paper, locust bean gum, cassava starch, and p-nitrophenyl b-D-xylopyranoside. The apparent Km value of the xylanase on soluble oat spelt xylan and insoluble oat spelt xylan was 7.3±0.236 and 60.2±6.788mg/ml, respectively. Thin-layer chromatography analysis showed that the xylanase hydrolyzed oat spelt xylan to yield mainly xylobiose and xylose as end products, but that it could not release xylose from the substrate xylobiose, suggesting that it is an endo-xylanase.

Directed evolution of a mesophilic fungal xylanase by fusion of a thermophilic bacterial carbohydrate-binding module

Bacterial GH10 xylanase usually contains carbohydrate-binding module (CBM) that binds to insoluble xylan. Differing from GH10 xylanase, we isolated a fungal GH11 xylanase containing a single catalytic domain from Aspergilus niger (XYN). The thermophilic CBM from Thermotoga maritima (TmCBM9-1_2) might increase the mesophilic XYN's thermo-activity and catalytic efficiency on insoluble xylan, we fused it with the TmCBM9-1_2 behaving as “hand” to grasp xylan actively. The chimeric xylanase XYN-TmCBM9-1_2 exhibited an optimal activity at pH4.2 and 49 °C, 2 °C higher than the thermo-activity of XYN. The chimeric xylanase's activity was 970.1 ± 5.8 U/mg on insoluble oat-spelt xylan, 4.2-fold of that on soluble birchwood xylan (228.1 ± 1.1 U/mg). In contrast, the XYN's activity was 226.9 ± 1.2 U/mg on insoluble oat-spelt xylan, only 40% of that on soluble birchwood xylan (567.2 ± 3.0 U/mg). Fusing with the TmCBM9-1_2 increased the XYN's property, indicating that we can direct to evolve a molecule's function through fusing domains of bacteria and fungi.

Purification and characterization of a high-thermostable <i>β</i>-xylanase from newly isolated <i>Thermomyces lanuginosus</i> THKU-49

Highly thermostable β-xylanase produced by newly isolated Thermomyces lanuginosus THKU-49 strain was purified in a four-step procedure involving ammonium sulfate precipitation and subsequent separation on a DEAE-Sepharose fast flow column, hydroxylapatite column, and Sephadex G-100 column, respectively. The enzyme purified to homogeneity had a specific activity of 552 U/mg protein and a molecular weight of 24.9 kDa. The optimal temperature of the purified xylanase was 70°C, and it was stable at temperatures up to 60°C at pH 6.0; the optimal pH was 5.0–7.0, and it was stable in the pH range 3.5–8.0 at 4°C. Xylanase activity was inhibited by Mn2+, Sn2+, and ethylenediaminetetraacetic acid. The xylanase showed a high activity towards soluble oat spelt xylan, but it exhibited low activity towards insoluble oat spelt xylan; no activity was found to carboxymethylcellulose, avicel, filter paper, locust bean gum, cassava starch, and p-nitrophenyl β-D-xylopyranoside. The apparent Km value of the xylanase on soluble oat spelt xylan and insoluble oat spelt xylan was 7.3 ± 0.236 and 60.2 ± 6.788 mg/ml, respectively. Thin-layer chromatography analysis showed that the xylanase hydrolyzed oat spelt xylan to yield mainly xylobiose and xylose as end products, but that it could not release xylose from the substrate xylobiose, suggesting that it is an endo-xylanase.

Purification and characterization of two sugarcane bagasse-absorbable thermophilic xylanases from the mesophilic Cellulomonas flavigena

We report the purification and characterization of two thermophilic xylanases from the mesophilic bacteria Cellulomonas flavigena grown on sugarcane bagasse (SCB) as the only carbon source. Extracellular xylanase activity produced by C. flavigena was found both free in the culture supernatant and associated with residual SCB. To identify some of the molecules responsible for the xylanase activity in the substrate-bound fraction, residual SCB was treated with 3 M guanidine hydrochloride and then with 6 M urea. Further analysis of the eluted material led to the identification of two xylanases Xyl36 (36 kDa) and Xyl53 (53 kDa). The pI for Xyl36 was 5.0, while the pI for Xyl53 was 4.5. Xyl36 had a Km value of 1.95 mg/ml, while Xyl53 had a Km value of 0.78 mg/ml. In addition to SCB, Xyl36 and Xyl53 were also able to bind to insoluble oat spelt xylan and Avicel, as shown by substrate-binding assays. Xyl36 and Xyl53 showed optimal activity at pH 6.5, and at optimal temperature 65 and 55 degrees C, respectively. Xyl36 and Xyl53 retained 24 and 35%, respectively, of their original activity after 8 h of incubation at their optimal temperature. As far as we know, this is the first study on the thermostability properties of purified xylanases from microorganisms belonging to the genus Cellulomonas.

Alteration of Bacillus subtilis XynA endoxylanase substrate selectivity by site-directed mutagenesis

Bacillus subtilis endoxylanase XynA was engineered by site-directed mutagenesis to alter its substrate selectivity, i.e. the ratio of its capacity to solubilise water-unextractable arabinoxylans (WU-AX) to its capacity to hydrolyse water-extractable AX (WE-AX). Four surface exposed aromatic residues were targeted for mutation to alanine, either individually (Y113 and W185) or jointly (F48, Y94, Y113 and W185), based on the general role of aromatic residues in the binding of endoxylanases to insoluble substrates and the hypothesis that they affect the activity of endoxylanases towards WU-AX. Determination of the substrate selectivity factors and WU-AX degradation profile of the wild type and mutant enzymes using an elaborate procedure requiring incubation of native wheat flour WU-AX and WE-AX with different enzyme levels showed that mutants W185A and 4mutA, the mutant containing all four mutations, displayed lower selectivity towards WU-AX than the wild type recombinant endoxylanase. Each of the endoxylanases had similar binding activity to wheat flour WU-AX and insoluble oat spelt xylan.

Comparative characterization of deletion derivatives of the modular xylanase XynA of Thermotoga maritima

The modular Xylanase XynA from Thermotoga maritima consists of five domains (A1-A2-B-C1-C2). Two similar N-terminal domains (A1-A2-) are family 22 carbohydrate-binding modules (CBMs), followed by the catalytic domain (-B-) belonging to glycoside hydrolase family 10, and the C-terminal domains (-C1-C2), which are members of family 9 of CBMs. The gradual deletion of the non-catalytic domains resulted in deletion derivatives (XynADeltaC; XynADeltaA1C and XynADeltaNC) with increased maximum activities (V (max)) at 75 degrees C, pH 6.2. Furthermore, these deletions led to a shift of the optimal NaCl concentration for xylan hydrolysis from 0.25 (XynA) to 0.5 M (XynADeltaNC). In the presence of the family 22 CBMs, the catalytic domain retained more activity in the acidic range of the pH spectrum than without these domains. In addition to the deletion derivatives of XynA, the N-terminal domains A1 and A2 were produced recombinantly, purified, and investigated in binding studies. For soluble xylan preparations, linear beta-1,4-glucans and mixed-linkage beta-1,3-1,4-glucans, only the A2 domain mediated binding, not the A1 domain, in accordance with previous observations. The XynA deletion enzymes lacking the C domains displayed low affinity also to hydroxyethylcellulose and carboxymethylcellulose. With insoluble oat spelt xylan and birchwood xylan as the binding substrates, the highest affinity was observed with XynADeltaC and the lowest affinity with XynADeltaNC. Although the domain A1 did not bind to soluble xylan preparations, the insoluble oat spelt xylan-binding data suggest that this domain does play a role in substrate binding in that it improves the binding to insoluble xylans.

Fusion of family VI cellulose binding domains to Bacillus halodurans xylanase increases its catalytic activity and substrate-binding capacity to insoluble xylan

A tandem repeat of the family VI cellulose binding domain (CBD) from Clostridium stercorarium xylanase (XylA) was fused at the carboxyl-terminus of Bacillus halodurans xylanase (XylA). B. halodurans XylA is an enzyme which is active in the alkaline region of pH and lacks a CBD. The constructed chimera was expressed in Escherichia coli, purified to homogeneity, and then subjected to detailed characterization. The chimeric enzyme displayed pH activity and stability profiles similar to those of the parental enzyme. The optimal temperature of the chimera was observed at 60 °C and the enzyme was stable up to 50 °C. Binding studies with insoluble polysaccharides indicated that the chimera had acquired an increased affinity for oat spelt xylan and acid-swollen cellulose. The bound chimeric enzyme was desorbed from insoluble substrates with sugars and soluble polysaccharides, indicating that the CBDs also possess an affinity for soluble sugars. Overall, the chimera displayed a higher level of hydrolytic activity toward insoluble oat spelt xylan than its parental enzyme and a similar level of activity toward soluble xylan.

A new xylanase from a Trichoderma harzianum strain.

A new xylanase (XYL2) was purified from solid-state cultures of Trichoderma harzianum strain C by ultrafiltration and gel filtration. SDS-PAGE of the xylanase showed an apparent homogeneity and molecular weight of 18 kDa. It had the highest activity at pH 5.0 and 45 degrees C and was stable at 50 degrees C and pH 5.0 up to 4 h xylanase. XYL2 had a low Km with insoluble oat spelt xylan as substrate. Compared to the amino acid composition of xylanases from Trichoderma spp, xylanase XYL2 presented a high content of glutamate/glutamine, phenylalanine and cysteine, and a low content of serine. Xylanase XYL2 improved the delignification and selectivity of unbleached hardwood kraft pulp.

Adsorption of Clostridium stercorarium xylanase A to insoluble xylan and the importance of the CBDs to xylan hydrolysis

Xylanase A (XynA) from Clostridium stercorarium, which consists of a catalytic domain and two family VI cellulose-binding domains (CBDs) each connected by a linker sequence, was found to bind to insoluble oat-spelt xylan as well as acid-swollen cellulose (ASC). Its adsorption to xylan was greatly dependent on the concentration of the phosphate buffer in the binding assay mixtures. The adsorption of XynA to insoluble xylan proceeded in accordance with a Langmuir-type isotherm. The Ka and [PX] max values of XynA for oat-spelt were 0.17 l/μmol and 5.0 μmol/g, respectively. Removal of the CBDs from XynA abolished the cellulose- and xylan-binding abilities of this enzyme and reduced the enzyme activity toward insoluble xylan but not soluble xylan. These results clearly indicated that the CBDs of XynA play an important role in the hydrolysis of insoluble xylan. Desorption of XynA from the ASC-XynA complex was effected by soluble saccharide solutions such as barley β-glucan, birchwood xylan, and glucose solutions as well as a cellobiose solution, indicating that the CBDs of XynA have an affinity for soluble saccharides in addition to insoluble polysaccharides.

Evaluation of catalytic activity and synergism between two xylanase isoenzymes in enzymic hydrolysis of two separate xylans in different states of solubility

Synergism of two xylanase (1,4) β- d-xylan xylanohydrolase, EC 3.2.1.8) isoenzymes from Streptomyces A451 was observed in enzymic hydrolysis of water-soluble and -insoluble fractions of two commercial xylan preparations. The reaction modes were determined qualitatively by TLC and quantitatively by GLC. With oat-spelt xylan as substrate, the total amount of xylose and xylobiose produced by using xylanase I and xylanase II in sequence was significantly higher than that by xylanase I or xylanase II individually, while the amounts of the relatively higher-molecular-weight oligosaccharides xylotriose to xylopentaose obtained by using isoxylanases individually were more than those obtained by using isoxylanases in sequence. A time-course study on the release of hydrolysis products over a 24-h period revealed that higher oligosaccharides were released initially followed by release of simpler sugars. A study of the hydrolysis of model oligosaccharides by crude enzyme preparation revealed a preference for higher oligosaccharides and demonstrated that xylopentaose was hydrolyzed to xylose, xylobiose, and xylotriose. A comparison was made of the hydrolysis of two different xylans, birchwood (hardwood) and oat-spelt (softwood), by crude xylanase. Product profiles showed that there were significant differences between the range and amounts of products released from the two different xylans. Studies on relative specific activities showed that xylanase II had overall lower specific activity toward various xylans than did xylanase I, and xylanase I, xylanase II, and crude xylanase showed lower specific activity toward insoluble xylan fractions than toward soluble xylan fractions. In addition, the reduction in specific activity was greater for insoluble birchwood than for insoluble oat-spelt xylan.

Purification and characterization of a complex-bound and a free ?-1,4-endoxylanase from the culture fluid of the anaerobic fungus Piromyces sp. strain E2

Two extracellular xylanases were purified to homogeneity from the culture filtrate of the anaerobic fungus Piromyces sp. strain E2 and their properties were studied. The enzymes are present in a High Molecular Mass complex (HMM-complex) and as free protein in nearly equal amounts. Both enzymes are most likely identical as all biochemical characteristics were identical. The molecular masses of the enzymes are 12.5 kDa, as estimated by gel chromatography and electrophoretic mobility. The activities of both enzymes are optimal at pH 6.0 and 50°C and the enzymes are stable up to 72h at 40°C. The enzymes have a pI of 9.1. The Km and Vmax, determined with xylan from oat spelts, were 3 mg · ml-1 and 2600 IU · mg-1 protein. The enzymes are active both on soluble and insoluble oat spelt xylan. The purified xylanases are inactive against Avicel, carboxymethylcellulose, p-nitrophenyl-β-d-glucoside, and p-nitrophenyl-β-d-xyloside. The products of the pure enzymes are predominantly xylo-oligosaccharides, indicating that the enzymes act as endoxylanases (1,4-β-d-xylan xylanohydrolases, EC 3.2.1.8).